Biosensor

ABSTRACT

A biosensor for quantifying a specific substance contained in a biological sample includes an electrically insulating base plate, a plurality of lead terminals formed on the base plate, a plurality of lead wires connected to the lead terminals, respectively, an electrode system including two working electrodes and one reference electrode connected to the lead wires, respectively, an insulating layer that insulates the electrodes, an enzyme reaction layer formed on the insulating layer and the electrodes, a spacer formed on the enzyme reaction layer so as to ensure a sufficient space that receives a sample, and a cover formed on the spacer. Further, the spacer has a sample introduction port opened at one side of the spacer, the cover has at least one slit for venting air existing in a sample receiving space defined by the spacer and the cover, and the slit extends to above the electrodes from one end of the cover.

This application claims priority under 35 U.S.C. § 371 fromPCT/KR02/01854, filed on Oct. 4, 2002 and under 35 U.S.C. § 119(a–d)from Korean patent application KR 2002/27971, filed on May 20, 2002.

TECHNICAL FIELD

The present invention relates to a sensor, and more specifically to abiosensor for quantifying a specific substance contained in a biologicalsample.

BACKGROUND ART

Generally, a biosensor comprises an electrically insulating base plate,an electrode system including a plurality of electrodes and formed onthe electrically insulating base plate using a screen printing method,and an enzyme reaction layer including a hydrophilic polymer,oxidoreductase and an electron acceptor and formed on the electrodesystem. When a sample liquid containing a substrate is dropped on theenzyme reaction layer of the biosensor, the enzyme reaction layer isdissolved to allow the substrate and enzyme to react with each other. Ata result, the substrate is oxidized, and then the electron acceptor isreduced. After such an enzyme reaction finishes, the concentration ofthe substrate in the sample liquid is determined from an oxidationcurrent obtained by electrochemically oxidizing the reduced electronacceptor.

As a biosensor for quantifying a specific substance contained in abiological sample using an electrochemical manner, a glucose sensor isknown. FIGS. 1 and 2 show a structure of the glucose sensor.

FIG. 1 is an exploded perspective view of a conventional biosensor inwhich a reaction layer is omitted. FIG. 2 is a longitudinal sectionalview of the biosensor shown in FIG. 1.

Referring to FIG. 1, silver paste is screen-printed on an electricallyinsulating base plate 1 to form leads 2 and 3 on a base plate 1.Conductive carbon paste containing a resin binder is then printed on thebase plate 1 to form an operating electrode 4 on the base plate 1. Theoperating electrode 4 is contacted with the lead 2. Electricallyinsulating paste is then printed on the base plate 1 to form aninsulating layer 6. The insulating layer 6 covers all portions exceptthe operating electrode 4 so that the exposed area of the operatingelectrode 4 is maintained to be constant. Conductive carbon pastecontaining the resin binder is printed on the base plate 1 to come intocontact with the lead 3 and thus to form a ring-shaped counter electrode5. Subsequently, on or near an electrode system including the operatingelectrode and the counter electrode, a reaction layer is formed.

The electrically insulating base plate 1 having the reaction layer and acover 9 having an air hole 11 are bonded to each other via a spacer 10,along dashed dot lines marked in FIG. 1, to manufacture a biosensor. Aslit 13 is formed at the spacer 10 to provide a sample supplying pathbetween the base plate and the cover. Referring to a longitudinalsectional view of the biosensor having the above-mentioned structure, ahydrophilic polymer layer 7 is disposed at the electrically insulatingbase plate 1 having the electrode system, and a reaction layer 8including enzymes and electron acceptors and a lecithin layer 8 a aredisposed on the hydrophilic polymer layer 7 in this order.

When a biological sample is contacted with an introduction port 12 ofthe biosensor having the above-mentioned structure, the biologicalsample fills the slit 13 acting as a sample receiving space, and at thesame time air in the sample receiving space is vented through an airhole 11 formed at the cover 9.

However, since the air hole 11 is formed at the upper part of thebiosensor, the biosensor is disadvantageous in terms of its handling dueto measurement errors caused by frequent contact with the air hole 11when using the biosensor. Considering the fact that the reactionprogresses immediately after the sample comes into contact with thereaction layer, it is important to rapidly absorb the sampleirrespective of viscosity of the sample. However, in the biosensorhaving the above-mentioned structure, since the air hole 11 for ventingair is arranged at the rear side of a sample introduction passage, rapidabsorption of the sample is limited. Such limited absorption of thesample causes measurement errors in biosensors that initiate themeasurement after checking whether or not the sample is completelyintroduced.

DISCLOSURE OF THE INVENTION

Therefore, the present invention has been made in view of the aboveproblems, and it is an object of the present invention to provide abiosensor capable of rapidly inducing absorption of a biological sample,thereby minimizing measurement errors caused by the biosensor.

It is another object of the present invention to provide a biosensorwhich can provide simplicity and convenience in handling the biosensorand accurately measure a reactive substance contained in a sample usingtwo working electrodes and one reference electrode.

To achieve the above objects, there is provided a biosensor comprising:an electrically insulating base plate 20; a plurality of lead terminals32 formed on the base plate 20; a plurality of lead wires 31 connectedto the lead terminals 32, respectively; an electrode system 40 includingtwo working electrodes 42 and 43 and one reference electrode 41connected to the lead wires 31, respectively; an insulating layer 50 forinsulating the electrodes 41, 42 and 43; an enzyme reaction layer 80formed on the insulating layer 50 and the electrodes 41, 42 and 43; aspacer 60 formed on the enzyme reaction layer 80 so as to ensure asufficient space for receiving a sample; and a cover 70 formed on thespacer 60,

wherein the spacer 60 has a sample introduction port 61 formed at oneside of the spacer 60, and the cover 70 has at least one slit 71 forventing air existing in a sample receiving space 62 defined by thespacer 60 and the cover 70, the slit 71 extending to above theelectrodes 41, 42 and 43 from one end of the cover 70.

In the biosensor according to the embodiment of the present invention,since the sample receiving space 62 is opened through the slit 71, abiological sample is rapidly introduced into the biosensor by virtue ofmaximized capillary effect.

In accordance with one aspect of the present invention, there isprovided a biosensor further including a curved groove 72 formed at oneend of the cover 70 toward the electrodes 41, 42 and 43, the slit 71being formed from a certain position of the curved groove 72 with apredetermined length. The biosensor having the above-mentioned structurecan collect a quantity of biological sample in the curved groove 72.

In accordance with the embodiment of the biosensor according to thepresent invention, the respective working electrodes 42 and 43 have thesame electrical resistance and area, and the reference electrode 41 isspaced at the same distance from the respective working electrodes 42and 43 and has an area above 1.5 times larger than the workingelectrodes 42 and 43.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanying drawing,in which:

FIG. 1 is an exploded perspective view of a conventional biosensor inwhich a reaction layer is omitted;

FIG. 2 is a longitudinal sectional view of the biosensor shown in FIG.1;

FIGS. 3 a and 3 b are a top view and a back view of a biosensoraccording to an embodiment of the present invention, respectively;

FIG. 4 is an exploded perspective view of a biosensor according to anembodiment of the present invention;

FIG. 5 is a cross-sectional view of a biosensor according to anembodiment of the present invention;

FIG. 6 is a front view of the biosensor when viewed from a direction A(shown by an arrow) in FIG. 5; and

FIGS. 7 a to 7 e are diagrams showing various electrode arrangements andpositions of the slit for venting air in a biosensor according to anembodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be explained in more detailthrough preferred embodiments, with reference to the accompanyingdrawings in such a manner that it may easily be carried out by a personhaving ordinary skill in the art.

FIGS. 3 a and 3 b are a top view and a back view of a biosensoraccording to an embodiment of the present invention, respectively.

Referring to FIG. 3 a, a plurality of lead terminals 31 corresponding tothe number of electrodes are formed at one end of an electricallyinsulating base plate 20. As shown in FIG. 3 b, the lead terminals 31are connected to electrodes 41, 42 and 43, respectively, formed at theother end of the electrically insulating base plate 20 throughrespective lead wires 32. As shown in FIG. 3 a, a slit 71 is formed at acover 70 of the biosensor S according to the embodiment of the presentinvention, and extends from a curved groove 72 formed at one end of thecover 70 toward the electrodes 41, 42 and 43 to at least above theelectrodes 41, 42 and 43. The slit 71, which will be discussed below,acts as an air-vent when a biological sample is introduced by thecapillary phenomenon.

The electrically insulating base plate 20 may be made of anon-conductive material such as polyethylene terephthalate, polyvinylchloride resin, polycarbonate resin, etc. A lead section 30 includingthe lead wires 32 and the lead terminals 31 may be formed in accordancewith a common method such as screen printing. In the embodiment of thepresent invention, the lead section 30 was formed by screen-printing asilver ink or a mixed ink of silver and silver chloride on the baseplate 20.

The structure of the biosensor S will now be explained in more detailwith reference to the exploded perspective view and cross-sectional viewof FIGS. 3 a and 3 b.

FIG. 4 is an exploded perspective view of the biosensor S according tothe embodiment of the present invention, and FIG. 5 is a cross-sectionalview of the biosensor according to the embodiment of the presentinvention.

Referring to FIG. 4, the biosensor S according to the embodiment of thepresent invention comprises the electrically insulating base plate 20,the base plate 20 having three lead terminals 32 formed on one end ofthe base plate 20, the lead terminals 32 being connected to threeelectrodes 41, 42 and 43, respectively, through the lead wires 31. Inthe electrodes 41, 42 and 43, a reference numeral 41 denotes a referenceelectrode, and reference numerals 42 and 43 denote working electrodes.These electrodes act to measure the amount of current generated duringoxidation and reduction of an electron acceptor included in an enzymereaction layer 80, which will be discussed below. The referenceelectrode 41 is arranged between the respective working electrodes 42and 43. This electrode arrangement makes it possible to measure theamounts of current in the reference electrode 41 and the respectiveworking electrodes 42 and 43. That is, the biosensor according to theembodiment of the present invention measures the amounts of currentbetween the first working electrode 43 and the reference electrode 41,and the second working electrode 42 and the reference electrode 41,compares the measured values and determines errors generated in themanufacture of the biosensor and the reaction with a substrate, therebyquantitatively obtaining the concentration of the substrate contained inthe biological sample with an increased accuracy.

In accordance with the embodiment of the biosensor according to thepresent invention, in order to measure the amounts of current in thereference electrode 41 and the respective working electrodes 42 and 43under the same electrochemical conditions, the respective workingelectrodes 42 and 43 must have the same electrical resistance and area,and the reference electrode 41 must be spaced at the same distance fromthe respective working electrodes 42 and 43. In addition, the area ofthe reference electrode (41) is preferably more than 1.5 times largerthan that of the working electrodes 42 and 43. Since the amounts ofcurrent generated in the reference electrode 41 and the respectiveworking electrodes 42 and 43 is proportional to the reactive area of theelectrodes, the relatively large area of the reference electrode 41 canreduce measurement errors between the reference electrode 41 and therespective working electrodes 42 and 43. The reference electrode 41 andthe working electrodes 42 and 43 are collectively referred to as “anelectrode system 40”. The electrode system 40 can be formed by a screenprinting method using a conductive carbon ink.

In order to insulate the electrodes 41, 42 and 43, an insulatingmaterial is partially coated on the electrodes 41, 42 and 43 except theupper portions of the electrodes 41, 42 and 43 to form an insulatinglayer 50, as shown in FIG. 5. As the insulating material, anon-conductive ink for screen printing or an ink for insulation can beused. The enzyme reaction layer 80 is formed on both the exposedportions of the electrodes 41, 42 and 43 and the insulating layer 50.The enzyme reaction layer 80 includes an enzyme reactive with theintroduced biological sample, and an electron acceptor.

The enzyme reaction layer 80 must include an enzyme reactive with asubstrate to be detected. That is, the enzyme reaction layer 80 caninclude different enzymes depending on the application of the biosensor.Examples of the enzymes and substrates are shown in Table 1 below. Asshown in Table 1, when the biosensor according to the embodiment of thepresent invention is a glucose sensor, the enzyme reaction layer 80includes glucose oxidase. When a blood sample as the biological sampleis introduced into the enzyme reaction layer 80 of the sensor, glucosein blood is oxidized by glucose oxidase, after which the glucose oxidaseis reduced. Herein, the electron acceptor included in the enzymereaction layer 80 oxidizes the glucose oxidase and then itself isreduced. The reduced electron acceptor loses its electrons on thesurface of the electrode, to which a constant voltage is applied, andthen is electrochemically reoxidized. Since the concentration of glucosein the blood sample is proportional to the amount of current generatedwhen the electron acceptor is oxidized, the concentration of glucose inthe blood sample can be measured by measuring the amount of currentthrough the lead terminals 32.

TABLE 1 Substrate Enzymes Glucose Glucose oxidase CholesterolCholesterol esterase Cholesterol oxidase Peroxidase CreatinineCreatininase Creatinase Sarcosine oxidase Lactate Lactate oxidase

On the other hand, in accordance with the biosensor S according to theembodiment of the present invention, a spacer 60 having a sampleintroduction port 61 for forming a sample receiving space is formed onthe enzyme reaction layer 80, and is sandwiched between the base plate20 and the cover 70. In order to form the sample receiving space 62between the cover 70 and the enzyme reaction layer 80 when the cover 70and the spacer 60 are bonded to each other, the spacer 60 must be higherthan the enzyme reaction layer 80 formed on the base plate 20. Thespacer 60 can be made of resin. In the embodiment of the presentinvention, a double-sided tape made of resin was used as the spacer 60.

In accordance with the biosensor S according to the embodiment of thepresent invention, the cover 70 is bonded to the spacer 60. At thistime, in order to vent air existing in the sample receiving space 62between the spacer 60 and the cover 70, the slit 71 is formed at thecover 70. For stable introduction of the biological sample into abovethe electrode 42, the slit 71 extends to at least above the electrodes41, 42 and 43 with a predetermined length.

In the biosensor S as shown in FIG. 5, the spacer 60 is bonded to theupper side of the insulating layer 50. However, the spacer 60 can bedirectly bonded to the base plate 20 instead of the insulating layer 50.

FIG. 6 is a front view of the biosensor when viewed from a direction Ain FIG. 5.

Referring to FIG. 6, the base plate 20 is placed at the bottom of thebiosensor S according to the embodiment of the present invention, theinsulating layer 50 insulates the electrodes 41, 42 and 43 formed on thebase plate 20, and the enzyme reaction layer 80 is formed on theinsulating layer 50. Since the spacer 60 is higher than the enzymereaction layer 80 and is placed around the electrodes 41, 42 and 43, thesample receiving space 62 is formed between the cover 70 and the enzymereaction layer 80. Since the slit 71 formed at the cover 70 acts as anair-vent, the biological sample is introduced into the sample receivingspace 62 by virtue of capillary phenomenon.

FIGS. 7 a to 7 e are diagrams showing various electrode arrangements andpositions of the slit 71 for venting air in the biosensor S according tothe embodiment of the present invention.

In the biosensor S according to the embodiment of the present invention,the electrode system 40 including the reference electrode 41 and theworking electrodes 42 and 43 can be formed as shown in FIG. 7 a. Thatis, the reference electrode 41 has an E-shape, and the respectiveworking electrodes 42 and 43 can be arranged at the upper and lowerportions of a horizontal axis B, which is formed at the center of thereference electrode 41. In this case, the respective working electrodes42 and 43 must also have the same electrical resistance and area, andthe reference electrode 41 must be spaced at the same distance from therespective working electrodes 42 and 43.

In accordance with another embodiment of the present invention, theelectrode system 40 can be formed as shown in FIG. 7 b. That is, whenthe sample receiving space 62 is placed in a vertical direction, thereference electrode 41 has an H-shape, and the respective workingelectrodes 42 and 43 can be arranged at the upper and lower portions ofa horizontal axis of the reference electrode 41. Also, the respectiveworking electrodes 42 and 43 must have the same electrical resistanceand area, and the reference electrode 41 must be spaced at the samedistance from the respective working electrodes 42 and 43 and have anarea above 1.5 times larger than the working electrodes 42 and 43.

In the electrode systems shown in FIGS. 7 a and 7 b, the slit 71 formedat the cover 70 extends to above the reference electrode 41 and theworking electrodes 42 and 43.

FIGS. 7 c to 7 e are diagrams showing various positions of the slit 71formed at the cover 70. For example, FIG. 7 c shows the slit 71 formedalong a length direction of the cover 70, FIG. 7 d shows a plurality ofslits 71 formed at the center of the cover 70, each slit being spaced ata predetermined interval, and FIG. 7 e shows the slit 71 formed alongthe right side of the cover 70. All slits 71 shown in FIGS. 7 c to 7 eextend to above the electrodes 41, 42 and 43 arranged on the base plate20, thereby stably introducing the biological sample into the upperportion of the electrodes 41, 42 and 43.

Operation of a glucose sensor as an example of the biosensor accordingto the embodiment of the present invention will be explained below.

Referring to FIG. 3 a, after the curved groove 72 of the glucose sensoris contacted with a blood sample, the blood sample is introduced intothe sample introduction port 61 by virtue of capillary phenomenon andsimultaneously air existing in the sample receiving space 62 is ventedthrough the slit 71 formed at the cover 70. Subsequently, the bloodsample is introduced into the first working electrode 43, the referenceelectrode 41 and the second working electrode 42 through the samplereceiving space 62, and the blood sample is then impregnated into theenzyme reaction layer 80. Glucose contained in the blood sampleenzymatically reacts with GOD, so that it is oxidized and simultaneouslythe GOD is reduced. The reduced GOD reacts with the electron acceptorand is reoxidized, after which the reoxidized GOD reacts with otherglucose not yet oxidized. The reduced electron acceptor migrates to thesurface of the electrode, to which a voltage is applied, and then loseselectrons at the surface to be electrochemically reoxidized. Thereafter,the electron acceptor continuously takes part in the above reaction. Thecurrent generated during the oxidation of the electron acceptor isproportional to the concentration of glucose in the blood sample.Accordingly, the concentration of glucose in the blood sample can bemore accurately and quantitatively obtained by measuring the amounts ofcurrent flowing in the first working electrode 43 and the referenceelectrode 41, and the second working electrode 42 and the referenceelectrode 41, respectively, and averaging the measured values.

To facilitate introduction of sample into the biosensor, the biosensoraccording to the present invention has the slit 71 formed at the cover,instead of an air hole. Accordingly, the biosensor according to thepresent invention can provide simplicity and convenience in handling thebiosensor. In addition, since the slit 71 extends from one end of thecover 70 to above the electrodes, the sample can be rapidly introducedinto the electrodes.

INDUSTRIAL APPLICABILITY

As described above, since the biosensor according to the presentinvention has an air-venting slit extending from one end of a cover toabove the electrodes, a biological sample can be rapidly introduced. Inaddition, measurement errors caused by the biosensor can be minimized bymeasuring the amounts of current flowing in the two electrodes and onereference electrode, and averaging the measured values. Furthermore,since the biosensor according to the present invention does not includea separate air hole for facilitate introduction of a sample into thebiosensor, it can provide simplicity and convenience in handling thebiosensor.

While the present invention has been described with regard to preferredembodiments thereof, the description is for illustrative purposes onlyand is not to be construed as limiting the scope of the invention.Various modifications and changes may be made by those skilled in theart without departing from the true scope of the invention as defined bythe appended claims.

1. A biosensor comprising: an electrically insulating base plate; aplurality of lead terminals formed on the base plate; a plurality oflead wires connected to the lead terminals, respectively; an electrodesystem including two working electrodes and one reference electrodeconnected to the lead wires, respectively; an insulating layer thatinsulates the electrodes; an enzyme reaction layer formed on theinsulating layer and the electrodes; a spacer formed on the enzymereaction layer so as to ensure a sufficient space to receive a sample;and a cover formed on the spacer, wherein the spacer has a sampleintroduction port opened at one side of the spacer, and the cover has atleast one slit that vents air existing in a sample receiving spacedefined by the spacer and the cover, the slit extending to above theelectrodes from one end of the cover.
 2. The biosensor as set forth inclaim 1, further including a curved groove formed at one end of thecover toward the electrodes, the slit being formed from a certainposition of the curved groove with a predetermined length.
 3. Thebiosensor as set forth in claim 2, wherein the slit is a plurality ofslits spaced at a predetermined interval along a length direction. 4.The biosensor as set forth in claim 2, wherein the respective workingelectrodes have the same electrical resistance and area, and thereference electrode is spaced at the same distance from the respectiveworking electrodes.
 5. The biosensor as set forth in claim 4, whereinthe reference electrode has an area more than 1.5 times larger than theworking electrodes.
 6. The biosensor as set forth in claim 1, whereinthe slit is a plurality of slits spaced at a predetermined intervalalong a length direction.
 7. The biosensor as set forth in claim 1,wherein the respective working electrodes have the same electricalresistance and area, and the reference electrode is spaced at the samedistance from the respective working electrodes.
 8. The biosensor as setforth in claim 7, wherein the reference electrode has an area more than1.5 times larger than the working electrodes.
 9. The biosensor as setforth in claim 1, wherein when the sample receiving space is placed in avertical direction, the reference electrode has an H-shape, and therespective working electrodes are arranged at the upper and lowerportions of a horizontal axis of the reference electrode.
 10. Thebiosensor as set forth in claim 9, wherein the respective workingelectrodes have the same electrical resistance and area, and thereference electrode is spaced at the same distance from the respectiveworking electrodes.
 11. The biosensor as set forth in claim 1, whereinwhen the sample receiving space is placed in a vertical direction, thereference electrode has an E-shape, and the respective workingelectrodes are arranged at the upper and lower portions of a horizontalaxis, which is formed at the center of the reference electrode.
 12. Thebiosensor as set forth in claim 11, wherein the respective workingelectrodes have the same electrical resistance and area, and thereference electrode is spaced at the same distance from the respectiveworking electrodes.